Macromolecular X-ray crystallography is an essential tool in modem drug discovery and molecular biology. Using X-ray crystallographic techniques, the three-dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic level resolution from X-ray diffraction data.
One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow large, well diffracting crystals of the macromolecule. As the techniques for collecting and analyzing X-ray diffraction data have become more rapid and automated, crystal growth has become a rate limiting step in the structure determination process.
Vapor diffusion is the most widely used technique for crystallization in modern macromolecular X-ray crystallography. In this technique, a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution. The resulting drop of liquid (containing macromolecule and dilute crystallization solution) is sealed in a chamber with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir of crystallization solvent either by hanging the drop from a glass cover slip or by sitting the drop on a pedestal above the level of the solvent in the reservoir. Over time, the crystallization drop and the reservoir solutions equilibrate via vapor diffusion of volatile chemical species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization of the macromolecule sample in the drop.
Liquid-liquid diffusion is another technique form, which include bringing a solution of the target molecule in contact with a liquid solvent also referred to as crystallization solvent.
Examples of devices and methods for Vapor diffusion and/or liquid-liquid diffusion techniques are disclosed in EP 1 119 654, U.S. Pat. No. 6,409,832, US 2003/61687, WO 2008/000276 and co-pending patent applications USP 61/154,527 and USP 61/169,987.
The process of growing biological macromolecule crystals remains, however, a highly empirical process. Macromolecular crystallization is dependent on a host of experimental parameters, including; pH, temperature, the concentration of salts in the crystallization drop, the concentration of the macromolecule to be crystallized, and the concentration of the precipitating agent (of which there are hundreds). In particular, the choice of solute conditions in which to grow crystals continues to be a matter for empirical determination. Consequently, the ability to rapidly and easily generate many crystallization trials is important in determining the ideal conditions for crystallization. Sets of preformulated crystallization solutions has been developed in order to rapidly and easily generate many crystallization trials e.g. as described in EP 1 119 654.
However there still is a need for new and improved sets of preformulated crystallization solutions.